The present invention relates to optical imaging with reduced background, and more specifically to systems and methods for directly measuring the amount of background noise and removing it from the detected signal.
Optical imaging is becoming more and more the method of choice for many measurement applications that demand high sensitivity and quantification. Recent scientific advances benefit from reliably detecting small and/or weak targets. In order to obtain reliable representations of such targets, it is desirable to use an imaging system that has low noise levels. Fluorescence imaging is one of the techniques that have significant capabilities to reliably achieve sensitive, quantitative measurements. With fluorescence imaging, the target to be imaged is illuminated by an optical signal having a first spectral content (excitation light), and a portion of such a signal is absorbed by at least part of the target and is re-emitted as an optical signal of a second spectral content (emission light). The emission light is then detected by a detection system as a measure of the properties of the target.
A fluorescence imaging system typically includes one or more sources and components that generate and deliver the excitation light to the target area. The system also includes components to collect light from the target area, separate the emission light from the excitation light, and deliver it to an optical sensor. One common method for separating the emission light from reflected and/or scattered excitation light is optical filtering. Various other methods are also used to achieve similar results. However, with all the techniques known to date, it is often difficult to completely prevent reflected and/or scattered excitation light from reaching the sensor. This adds an amount of non-fluorescence signal to the emission signal which, in turn, results in a non-accurate measurement of target properties such as quantity of fluorescence material. This is one type of optical background noise. Another known type of optical background is auto-fluorescence which results from non-target elements in the system absorbing a portion of the excitation light and re-emitting it as fluorescence, a portion of its content being within the second spectral range. Examples of components that can generate auto-fluorescence background noise include the media where the target resides, optical filters, and lenses. Yet another source of optical background noise is light generated by sources other than the excitation light sources that makes its way towards the sensor. There are also a number of other non-optical background sources, such as dark signals generated by the sensor itself and the electronics that drive it. They, too, cause an increase in the background noise and if not eliminated or reduced, limit the performance of the imaging system.
There are a number of techniques and implementations for improving the sensitivity of fluorescence imaging systems (see, e.g., U.S. Pat. No. 6,921,908; U.S. Pat. No. 6,495,812; U.S. Pat. No. 7,286,232; and U.S. patent application Ser. No. 12/785,308, the contents of each of which are hereby incorporated by reference for all purposes). These techniques range from hardware configurations to software processing of acquired images. Methods that rely more on hardware techniques are often preferred because they aim at the root-cause of the problems not at their symptoms. Within the hardware solutions, the most efficient are those that leverage any differences between the optical properties of target fluorescence emission and background noise to favorably select the former. And, as is well known in the optical imaging industry, techniques that work best for one type of background noise may not work well for others. There are also techniques that address more than one type of background noise in one design, but there still is a need for a more general way to eliminate or reduce the background noise irrespective of its origin or type.
Therefore it is desirable to provide systems and methods that overcome the above and other problems.